Chronic myelogenous leukemia (CML) accounts for 20% of all cases of leukemia and carries a death rate of 1.5 per 100,000 population (Lichtman, chronic Myelogenous Leukemia and Related Disorders in Hematology, Williams (ed.) 4th Ed., McGraw Hill, New York, N.Y. (1990)). In 1960, Nowell and Hungerford discovered that the Philadelphia chromosome (Ph+) was consistently associated with CML (reviewed in Rosson and Reddy, Mutation Res., 195, 231-243 (1988)). Molecular studies have demonstrated that during the formation of the Philadelphia chromosome, a portion of the c-abl gene is translocated from chromosome 9q34 to chromosome 22q11, resulting in the formation of a chimeric gene consisting of sequences derived from the BCR and ABL loci (Heisterkamp et al., Nature, 306, 239 (1983); Heisterkamp et al., Nature, 315, 758 (1985)). This translocation is detectable in over 95% of patients with CML. A significant portion of acute lymphocytic leukemia (ALL) patients also carry the Ph.sup.1 chromosome.
The fusion gene, named bcr-abl, transcribes a chimeric mRNA of 8.5 kb which is translated to a p210.sup.bcr-abl fusion protein with altered tyrosine kinase activity (Konopka and Witte, Mol. Cell Biol., 5, 3116-3123 (1985); Kurzrock et al., N. Engl. J. Med., 319, 990 (1988)). The p210.sup.bcr-abl protein has been shown to transform myeloid precursor cells in vitro (McLaughlin et al., Proc. Natl. Acad. Sci. USA, 84, 8558 (1987)). In addition, infecting murine bone marrow stem cells with a retrovirus containing the bcr-abl gene produces a disorder similar to human CML in mice (Daley et al., Science, 247, 824 (1990); Voncken et al., Blood, 79, 1029-1036 (1992)
It has been shown using antisense molecules that interrupting the bcr-abl transforming signal can inhibit the cell growth of isolated blast cells from CML patients (Szczylik et al., Science, 253, 562 (1991); WO 92/22303 (1992)).
In the past decade, the use of antisense nucleic acid sequences to block the function of a mRNA has been developed as a strategy to inhibit viral and malignant diseases (Weintraub, Sci. Amer. 262, 34-40 (1990)). More recently, it has been shown that specific RNA sequences have catalytic activity (Kruger et al., Cell 81, 147-157 (1982)). These molecules are termed "ribozymes". Several different catalytic sequences have been described with a common need for divalent cations, which cause nonhydrolytic transesterification of specific RNA target regions (Symons, Annu. Rev. Biochem. 61, 641-671 (1992)). These catalytic sequences have been modified to create antisense molecules which bind to and cleave specific target RNA molecules (Zaug et al., Nature, 324, 4229-4233 (1986); Uhlenbeck, Nature 328, 596-600 (1987)).
Snyder et al., Blood 82,600-605 (1993) have shown that liposome vectors containing a single-unit DNA-RNA hybrid ribozyme can inhibit cr-abl gene expression and cell growth in a Ph-positive cell line. Other reports of ribozyme-mediated inhibition of cr-abl gene expression include WO 93/03141 (1993), and WO 92/00080 (1992) and the corresponding Reddy et al., U.S. Pat. No. 5,246,921. The "hammerhead" ribozyme has been developed into a targeted ribozyme, employing a catalytic "hammerhead" domain and flanking oligonucleotides that specifically bind to target sequences (Walbot, Nature 334, 585-591 (1988)). Cleavage occurs 3' to a GUX triplet where X can be C, U, or A (Guerrier-Takada et al., Cell 35, 848-852 (1983); Cech & Bass, Annu. Rev. Biochem. 55, 599-629 (1986); Symons, Trends Biochem Sci. 14, 445-50 (1989)). Any GUX sequence can be targeted by the appropriate design of the flanking oligonucleotide sequence.
There are no conventional therapies including chemotherapy, .sup.32 P-treatment and splenic irradiation, that have resulted in cures in CML (Lichtman, Chronic Myelogenous Leukemia and Related Disorders in Hematology, W. J. Williams, Ed., 4th Edition, McGraw Hill, New York, N.Y. (1990)). Five to 15% of patients receiving alpha-interferon therapy may suppress the expression of the Ph-positive clone in CML (Talpaz et al., N. Engl. J. Med. 314, 1865-1859 (1986). However, the ability of interferon to cure CML is not proven. Allogeneic bone marrow transplant (BMT), using HLA identical siblings, following myeloablative chemo-radiotherapy is curative in up to 85% of carefully selected patients in chronic phase CML (Goldman et al., N. Engl. J. Med. 314, 202-207 (1986); Thomas et al., Amer. Intern. Med.104, 155-166 (1986); Marks et al., Br. J. Haematol. 81, 383-390 (1992)). In CML patients transplanted with an identical twin following high dose myeloablative therapy, the cure rate is 50%. This difference is due to a graft-versus-leukemia (GVL) effect and is enhanced by the presence of chronic graft versus host disease (GVHD). T-cell depletion of donor marrow reduces the GVL effect, the incidence of GVHD, as well as the cure rate (Gale et al., Bone Marrow Transplantation 9, 83-85 (1992)). Collectively, these data show that high dose therapy given early in the course of CML is potentially curative in 50% of patients. The additional curative effects of allogeneic BMT are due to a GVL effect.
Unfortunately, less than 30% of CML patients will have a normal allogeneic HLA matched donor. Current use of matched unrelated donors has resulted in high mortality due to GVHD and infections (Marks et al., Ann. Int. Med. 119, 207-214 (1993)). Thus, the development of an autologous marrow transplant program using Ph-negative stem cells, would provide an alternative for patients without other curative options. An autologous BMT would avoid GVHD and should be curative in up to 50% of patients, provided the marrow is purged free of CML stem cells. In preparation for autologous marrow infusion, marrow cells are harvested from the affected individual, are "purged" of leukemia cells by chemical agents, and returned to the patient following extensive chemotherapy or total body radiation.
Thus far, autologous BMT has not been successful in CML because Ph-positive stem cells are invariably reinfused into the patient (Reiffers et al., British aematology 77, 339-345 (1991)). In an attempt to provide Ph-negative stem cells for autologous BMT, chemotherapeutic purging (Silvestri et al., Int. J. Cell Cloning 9, 474-490 (1990)), interferon purging (McGlave et al., Bone Marrow Transplantation 6, 115-120 (1990)), long term bone marrow cultures (Barnett et al., Bone Marrow Transplantation 4, 345-351 (1989)), and CD34 stem cell selection (Verfaillie et al., Blood 79, 1008-1010 (1992)) have all been attempted and may result in transient Ph-negative hematopoiesis. Using antisense molecules, Szczylik et al., Science, 253, 562 (1991) and WO 92/22303 (1992) inhibited the cell growth of isolated blasts from CML patients.
While the presently known bcr-abl ribozymes (Snyder et al., Blood 82, 600-605 (1993) and WO 93/03141 (1993); Reddy et al., WO 92/00080 and U.S. Pat. No. 5,246,921) are promising, there is a need for bcr-abl ribozymes with increased catalytic potential for oncogenic bcr-ablmRNA, without causing substantial cleavage of normal c-abl and bcr transcripts. There is also a need for bcr-abl ribozyme vectors having improved uptake.